Lecture 15: Major CNS NT Synapses and Their Receptors:

Neurons in the Human CNS

• Contains ~86 ×10⁹ neurons

  • Glutamate (glutamatergic):↑70%;60 x 10⁹

  • GABA (GABAergic):  ↑30%; 26 x 10⁹

    • Use GABA/ glycine as a NT

  • “Neuromodulators”: <0.1%

    • Dopamine neurons: 400-600 x 10³

    • 5-HT (serotonin) neruons: 300 x 10³

    • ACh (Nucleus Basalis Meynert) neurons: 200 x 10³

    • Noradrenaline: 20-50 x 10³ 

• Important systems that are targted neuropharamcologically to modulate the nervous system

• Synapses per neuron in the brain - Between 10¹⁴ and 10¹⁵ (100 trillion - 1 quadrillion)

Grey Type 1 Synapse

• Asymmetrical structure

  • Round vesicles

  • Large active zone – dense in protein and release machinery

  • Prominent and dense ECM between the pre-synaptic and post-synaptic membrane

  • The postsynaptic membrane is protein-dense

• Excitatory” - associated with L-glutamatergic synapse markers

  • (antibodies to L-glutamate) – label the synapses exclusively

Why is It Hard to Find Synapses for Neuromodulatory Substances

• They are released through volume transmission → released into the extracellular space and influence the surroundings

Grey Type 2 Synapse

• Symmetrical structure

  • Flattened vesicles – contain GABA/ glycine

  • Less prominent active zone

  • Lower postsynaptic densities

  • Less prominent extracellular matrix/ base membrane

• “Inhibitory” - associated with GABAergic and glycinergic

  • Dependent on area of the NS

  • synapse markers (antibodies to GABA)

Excitatory CNS Neurotransmitters

• ubiquitous (“everywhere”)

• act between the cerebral cortex → spinal cord

• Make up 60-70% of all synapses

Inhibitory CNS Neurotransmitters:

• γ-Aminobutyric acid (GABA) - cerebral cortex → brain stem

• Glycine - brain stem → spinal cord

• 20-30% of all synapses

Ionotropic Receptors

• Both excitatory and inhibitory neurotransmitters act on these receptors.

• They modify the membrane potential by allowing ions to move across the membrane.

Metabotropic Receptors

• Involve activation of G-proteins.

• G-protein activation influences various effector targets, such as ion channels.

• These ion channels modify cell excitability or influence presynaptic release processes.

Excitatory Neurotransmission

• Mediated by glutamate

• Glutamate has an excitatory effect when applied to neurons, resulting in the depolarisation of the membrane and AP firing

• Applicated to a VC neurons - generation of an inward current, that drives depolarisation

• These responses are said to be mediated by the ionotropic receptors

Voltage-dependence of fast excitatory neurotransmission

• Activation of a synapse generates a triangular-shaped depolarisation with a rapid rise, peak, and slow decay as the neurotransmitter diffuses away.

• Glutamatergic neurotransmission involves two main receptor types (AMPA & NMDA) with voltage dependence affecting the shape of the EPSP.

• Graphs: Membrane potential typically at -80mV but can change due to synaptic activity.

• Control condition: No antagonists; EPSPs show a rapid rise with a longer decay if the duration is prolonged.

• AMPA (green): EPSPs mediated by AMPA receptors in the presence of NMDA antagonists (e.g., D-AP5).

• NMDA (orange): EPSPs mediated by NMDA receptors in the presence of AMPA antagonists (e.g., NBQX).

• Voltage-dependence: NMDA receptors have a larger role at depolarized membrane potentials, contributing more to EPSPs at higher membrane potentials.

Explain the Graph:

• Control condition: No antagonists; EPSPs show a rapid rise with a longer decay if the duration is prolonged.

• AMPA (green): EPSPs mediated by AMPA receptors in the presence of NMDA antagonists (e.g., D-AP5).

• NMDA (orange): EPSPs mediated by NMDA receptors in the presence of AMPA antagonists (e.g., NBQX).

• Voltage-dependence: NMDA receptors have a larger role at depolarised membrane potentials e.g. -40mv, contributing more to EPSPs at higher membrane potentials.

AMPA Receptor Electrophysiology

• At 0mV: No response seen (ions at equilibrium potential).

• Further depolarisation: Voltage responses reverse and become hyperpolarized.

• Under voltage clamp (below 0mV):

  • Inward currents support EPSPs.

  • At more depolarized potentials, reversal is seen (no net current during EPSP generation).

  • At positive depolarized levels, outward current generated → move away from E_ion

• It is a non-specific cation channel (shown by GHK equation).

• At 0mV: Inward and outward ion fluxes balance (net current = 0).

• Linear IV plot: No voltage dependence (Ohmic behavior when activated).

  • linear conformist channel when activated

Voltage Dependence of NMDA-Receptors

• I/V Relationship :

  • Depolarisation to -30mV results in a larger EPSC than at resting membrane potential (-70mV).

  • Non-linear response — receptors are “deviant” or “non-conformist” and are targets for drugs like ketamine and ethanol.

• IV Relationship Distortion:

  • Above -30mV, NMDA receptor responses resemble AMPA receptors.

  • Below -30mV: the response becomes non-linear, deviating from Ohm's law (less current, indicating an interference with ion flow at hyperpolarised potentials).

  • Current through NMDA receptors is linear around 0mV, and the ion flow reverses around this point, following Ohm's law in this range.

Effect of Removing or Decreasing Mg2+ From Bathing Solution of NDMA Receptors on Current Relationship

• ohmic relationship seen - linear plo

Mg²⁺ Block Of NMDA-Receptors

• Mg2+ acts as a channel blocker by becoming trapped in the open NMDA receptor channel at hyperpolarised potentials, preventing ion flow.

  • block occurs at hyperpolarised potentials after ligand binding and receptor activation to block ion flow.

• At depolarised potentials, the electrostatic Mg²⁺ block weakens, allowing ions to flow through the channel, resulting in linear (ohmic) behaviour.

Similarity in NMDA and K⁺ Channels

• Evolutionarily similar as both are tetramers.

• However, Mg²⁺ blockage occurs in the opposite direction for NMDA receptors compared to K⁺ channels.

Ionotropic Glutamate Receptors (iGluRs)

• The receptor subunits are categorised by sequence homology.

• The closer they are on the homology tree, the more similar their amino acid sequences

• These functional receptors are

  • tetramers (4 subunits) with 4 agonist-binding sites.

  • Homomeric or heteromeric: Difficult to identify subunits in situ.

  • Non-selective cation channels (K+, Na+, Ca2+) with Eion ~ 0mV.

Types of iGluRs: AMPA Receptors

• GluA1-4: 4 different subunits

  • Receptor can be homomeric or heteromeric - difficult to identify subunits in situ

• Named after its agonist agonist

Types of iGluRs: NMDA Receptors

• GluN1-2 → Complex subunits with dual agonism.

  • GluN1: Glycine binding site.

  • GluN2: Glutamate binding site.

  • Requires both glycine and glutamate for activation.

• Heteromeric

Types of iGluRs: Kinate Receptors

• GluK1-5 subunits

  • Receptors may be homomeric or heteromeric - difficult to identify in situ

• Named after agonists

• limited distribution.

Postsynaptic Electrophysiology: Group I mGluRs

• Glutamate acts on inotropic and metabotropic receptors (mGluRs) at the postsynaptic terminal.

• Agonist: Dihydroxyphenylglycine (DHPG)

  • Selective for mGluRs; can cause membrane depolarisation and AP firing.

  • TTX (Tetrodotoxin) is used to block Na+ channels to assess excitation and block AP generation.

  • DHPG modulates ionotropic receptors (e.g., NMDA receptors) by increasing depolarisation and enhancing excitability.

• Three classes of metabotropic glutamate receptors are involved in post-synaptic effects and regulation of excitatory neurotransmission.

Pre-synaptic Electrophysiology Group II mGLuRs

• The receeptor regulates the function and causes a depression of release

• mGluR agonist: DCG-IV

  • Depresses synaptic transmission through a pre-synaptic action

  • Reduces the amplitude of responses

  • Affects the mossy fibre pathway (dentate gyrus → area CA3)

Pre-synaptic Electrophysiology Group III mGLuRs

• The receptor regulates the function and causes a depression of release

• mGluR agonist: L-aminophosphonobutyrate (L-AP4)

  • Depresses synaptic transmission through a pre-synaptic action

  • Reduces the amplitude of responses

Metabotropic Glutamate Receptors (mGluRs)

• The receptor subunits are categorised by sequence homology.

• The closer they are on the homology tree, the more similar their amino acid sequences

• There are 8 subtypes of receptor

• 3 Principal receptor groups include

  • Group 1: Postsynaptic

  • Groups 2 and 3: Presynaptic

• These receptors function as homodimers and consist of:

  • Ligand binding domain: Recognizes glutamate.

  • 7 TM domains,

  • C-terminal intracellular loop.

  • Dimer formation creates a functional receptor that activates G-proteins.

Effector Targets of Group I mGlu-Rs (Post Synaptic)

• These mGluRs directly modify channel function via activation of PKC via the αq11 subunit of a G-protein.

• This activation increases PLC (phospholipase C), generating DAG and IP3, which further increase PKC.

• PKC phosphorylation of Tandem 2-pore domain K+ channels (K2P) results in their closure → depolarisation.

• NMDA-R current is enhanced by PKC phosphorylation, leading to increased NMDA receptor-mediated currents.

• Effect: Enhanced excitability and increased responsiveness to NMDA signaling.

Effector Targets of Group II & III mGlu-Rs (Pre-synaptic)

• Target Ca2+ channels, decreasing their function

  • Target P/Q- and N-type (CaV2.1-2) Ca2+ channels, reducing neurotransmitter release.

• Mechanisms:

  • Direct: G-protein βγ dimer interacts with Ca2+ channels to inhibit function.

  • Indirect: αi subunit → ↓ cAMP → ↓ PKA → enhances phosphatase activity → dephosphorylation of Ca2+ channels.

• Result:

  • Decreased likelihood of neurotransmitter release and synaptic responsiveness → decrease in excitabliity

  • Modified excitability due to shift in balance towards dephosphorylation of Ca2+ channels.

Key events at a glutamatergic synapse

1. Early fast EPSP: Mediated by ionotropic receptors (AMPA or AMPA+NMDA), dependent on voltage.

2. Delayed/late EPSP: Initiated by the closure of K+ channels due to activation of postsynaptic mGluRs.

3. K+ channel closure: Mediated by the B-y dimer; phosphorylation plays a role.

4. NMDA upregulation: Phosphorylation increases NMDA channel activity, leading to augmented responses.

5. Glutamate releasectivates ionotropic receptors on the postsynaptic density, opposite the release site.

6. mGluRs outside the postsynaptic density: Can modify excitability.

7. Autoreceptors on presynaptic terminal: Modulate NT release; feedback loop when presynaptic terminal is repeatedly activated.

Inhibitory Neurotransmission

• GABA: Primary _______ neurotransmitter in the forebrain.

• Glycine: Major ______ neurotransmitter in the spinal cord and brainstem.

• Both act at ionotropic receptors to hyperpolarise the membrane potential, preventing neuron firing

• GABA acts at a subset of metabotropic receptors

• These neurotransmitters hyperpolarise the membrane potential, inhibiting an active neuron from firing.

• Hyperpolarisation occurs when activation of receptors causes an outward current, moving the MP toward a more negative, hyperpolarised state

Ionotropic GABA/Glycine Receptor Electrophysiology

• These channels are anion-selective channels - Cl^-; E_ion ~ -70mV

• Above -70mV: hyperpolarisation that increases as the membrane potential moves further from E_ion

  • Outward current → upward deflection → Inhibitory Post-Synaptic Potential (IPSP)

• Below -70mV:

  • Activation of receptors can lead to depolarisation (moves MP toward -70mV)

  • Inward current → downward deflection

  • Potential for excitatory effect (debated)

• Effect is primarily inhibitory → break of activity, prevents AP firing

• Outward current → chloride influx (Cl-) at hyperpolarised states

  • Inward current → chloride efflux

• IV plot characteristics:

  • Linear, ohmic relationship

  • Constant conductance (channels always open

GABA-A receptors

• Ionotropic receptor: Part of the same gene superfamily as glycine receptors.

• Structure: Pentameric (five subunits), typically composed of 2 α-, 2 β-, and 1 γ-subunit.

• Pharmacology: Wide variety due to different subunits (e.g., α- and β- subunit interface where GABA binds).

  • Different sensitivities do different neuromodualtors

• Function: Opens Cl⁻ channels, producing an inhibitory effect.

• Drugs: Target the α-β interface to modify GABA activation.

Glycine Receptors

• Ionotropic receptor: Related to GABA-A receptors, part of the same gene superfamily.

• Structure: Pentameric, composed of alternating A- and B-subunits (B-A-B-A-B stoichiometry ).

• Function: Inhibitory, opens Cl⁻ channels to exert effects.

• Shares functional characteristics with GABA-A receptors.

GABA-C Receptors

• Ionotropic receptor: Structurally distinct from GABA-A receptors.

• Subunits: Composed of Rho subunits.

• Function: Produces inhibitory effects in various CNS regions by opening Cl⁻ channels.

• Distinct subunit composition compared to other ionotropic receptors

GABA-B Post Synaptic Electrophysiology

• Agonist: Baclofen

• Results in hyperpolarisation with a longer time course

  • Due to the activation of metabotropic receptors

  • Forms part of the IPSP (Inhibitory Postsynaptic Potential)

• Effect of GABA-B antagonist:

  • Late phase of IPSP disappears

  • EPSP shape speeds up

• This shows that GABA-B receptor-mediated hyperpolarization contributes to the inhibitory synaptic response.

2 Phases of IPSPs

• Fast phase

• Slow phase

GABA-B Pre-Synaptic Electrophysiology

• Inhibition of IPSP

  • Reversible depression observed with baclofen application and its subsequent removal

• Regulates of glutamatergic function at glutamatergic terminals

  • Baclofen (agonist) reduces the size of glutamate EPSP

• Receptors regulate both inhibitory and excitatory synaptic transmission across a wide range.

Metabotropic GABA-B Receptors

• Composed of 2 different subunits that form a complex arrangement → can’t function independently

  • GABA_B1-R subunits: agonist binding site for GABA

  • GABA_B2-R subunits: signals to G-proteins

• Receptors form a dimer of heterodimers, requiring both subunits to be functional

  • 2 agonists binding sites must associate with the effector subunits to activate the G-protein

• Affect target similarly to metabotropic glutamate receptors:

  • Target K+ ion channels (post-synaptic effects, membrane potential regulation).

  • Target Ca2+ channels (pre-synaptic effects, modulation of neurotransmitter release).

GABA-B Receptors - Post Synaptic Effector Targets

• The receptors are directly liked to the post-synaptic Kir channels via the B-y dimer, following the activation of the G-protein and receptor

• Activation of G-protein coupled to inward rectifier K+ channels (GIRKS) → Kir 3.1-4 and upregulates its functionality – greater movement of K+ ions and a greater effect on MP

  • increase channel function

• Postsynaptic → hyperpolarization

  Reduced excitabilit

GABA-B Receptors: Pre Synaptic Effector Targets

• decrease pre-synaptic calcium channel function.

• Mechanisms:

  • Direct: βγ dimer of G-protein interacts with P/Q- and N-type Ca²⁺ channels (CaV2.1 & CaV2.2) → ↓ Ca²⁺ entry → ↓ neurotransmitter release.

  • Indirect: αi subunit of G-protein ↓ cAMP → ↓ PKA → dephosphorylation of Ca²⁺ channels → ↓ channel functionality.

    • Balance between protein kinases and phosphatases shifts in favour of dephosphorylation of Ca²⁺ channels and reduces their functionality

• Result: Decreased channel activity reduces liklihood of neurotransmitter release, leading to inhibitory effects on neurotransmission and modifying excitability.

Key Mechanisms in Inhibitory Neurotransmission

• Early IPSP: Mediated by GABA-A receptors.

• Late IPSP: Mediated by GABA-B receptors, which are coupled to inward rectifier channels causing inhibition.

• Pre-synaptic autoreceptors: Located on inhibitory presynaptic terminals, reduce neurotransmitter release by inhibiting the release process.

• GABA can escape the synapse and inhibit neighbouring excitatory synapses, reducing glutamatergic neurotransmission.

Interplay between excitatory and inhibitory influences in major NT synapses in the CNS

• EPSP: Depolarises the membrane. If large enough, it reaches the threshold and triggers an action potential (AP).

• IPSP: Hyperpolarises the membrane, pushing the membrane potential closer to -70mV, inhibiting action potential generation.

• Combined Activation: Excitatory and inhibitory signals together regulate the neuron’s approach to the firing threshold, balancing excitation and inhibition.

  • inhibitory inputs can regulate the excitatory inputs and modify the neurons approach to threshold

Two ways interneurons modify excitatory function:

1. Direct influence: Excitatory input on a principal neuron can trigger an EPSP and activate an inhibitory interneuron, to produce an IPSP.

2. Feedback influence: Excitatory input. can activate the principal neuron, which can influence inhibitory interneurons to feedback and modify subsequent excitatory responses and neurons.

• Feedforward and feedback inhibition: Present to regulate neuronal network excitability.

Prinicpal Cells in Corical Circuits

• Principle cells are wired to have reciprocal excitatory connections as well as feedforward and feedback inhibitory connections

• Intact inhibition: excitability of the system is low, with activation of excitatory inputs generating a modest response

• Removal of inhibition: excitation propagates in the cortical circuit, between principal cells, allowing for a long-lasting excitatory response which is sustained over a period of time (in response to a single input)

Importance of Inhibition in Cortical Excitabilitiy

• It controls cortical excitability.

• Without it, overexcitation can occur, leading to epilepsy.

• In particular cortical areas and the nervous system, inhibition is crucial for preventing sustained excitatory circuits that can trigger seizures.

• Competitive antagonists of GABAA receptors (e.g., Bicuculline) and Glycine receptors (e.g., Strychnine) block inhibition, leading to convulsions and seizures due to overexcitation.